2 "-Ethyl-furanoflavone Derivatives from the Stems of Cassia fistula and their Cytotoxicity

Duas novas 2"-etil-furanoflavonas chamadas fistulaflavonas A e B juntamente com seis furanoflavonas conhecidas foram isoladas das hastes da Cassia fistula. As estruturas foram elucidadas por métodos espectroscópicos, incluindo as técnicas de NMR 1D e 2D e espectrometria de massa com ionização por electrospray de alta resolução (HRESIMS), e comparação com dados da literatura. Todos os compostos foram avaliados com relação a citotoxicidade para cinco linhas de células tumorais humanas. Um dos compostos mostrou potente citotoxicidade contra células SHSY5Y e MCF7, com valores de IC50 de 2,6 e 2,7 μmol L , respectivamente.


Introduction
Cassia fistula L., (Leguminosae) is an ornamental tree with beautiful yellow flowers.This plant can be found in various countries in Asia, South Africa, Mexico, China, West Indies, East Africa and Brazil. 1 In China, it has been widely used as traditional Chinese medicine for treatment of diarrhea, gastritis, ringworm and fungal skin infections. 2,3revious phytochemical studies of C. fistula have shown the presence of anthraquinones, 4,5 steroids, 6 chromones 7,8 and flavonol derivatives. 9With the aim at searching for new natural compounds from medicinal plants, the stems of C. fistula were investigated and two new 2"-ethylfuranoflavones (1-2) and six known furanoflavones (3-8)  were isolated.The structures of the isolated compounds were established by spectroscopic methods including extensive 1D ( 1 H, 13 C and DEPT) and 2D (one-bond HSQC and long-range HMBC) NMR techniques and high resolution electrospray ionization mass spectrometry (HRESIMS), and by comparison with literature data.This work deals with the isolation, structural characterization of these compounds and their cytotoxicity against five human tumor cell lines.

Plant material
The stems of Cassia fistula L. (Leguminosae) were collected at Xishuangbangna Prefecture, Yunnan Province, People's Republic of China, in September 2010.The identification of the plant material was verified by Dr. Yuan N., Kunming Institute of Botany, Chinese Academy of Sciences.A voucher specimen (YNNU 10-9-25) was deposited in our Laboratory.

Cytotoxicity assay
The cytotoxicity tests were performed against NB4, A549, SHSY5Y, PC3 and MCF7 tumor cells by MTT (yellow dye 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide) assay using Taxol ® as the positive control. 18Firstly, 2500 cells suspended in 100 μL MEM medium were seeded, in a 96-well plate.After 24 h incubation, fresh medium containing various concentrations of each compound were added into the 96-well plate to replace the old medium.The OD 595 values of the control groups at 0 and 72 h together with the compound treated groups at 72 h from the MTT assay were measured using a plate reader.
4, s) and C-3" (d C 101.5, d), of H-3" (6.84, s) with C-4" (d C 21.4, t) confirmed the ethyl group located at C-2".Cross peaks (Figure 2) of H-3" (d H 6.84, s) to C-7 (d C 159.3, s), C-8 (d C 111.5, s) and C-9 (d C 148.4, s) were also observed in compound 1.This allowed us to conclude that the 2"-ethylfurano moiety was fused in an angular manner on the aromatic ring at positions C-7 and C-8.The HMBC correlations of the methoxy protons (d H 3.85, s) with C-4' (160.7,s) revealed that the methoxy group should be located at C-4'.Two chelated hydroxy groups were assigned to C-3 and C-5 on the basis of HMBC correlations between the hydroxy proton (d H 10.83, brs) and C-2 (d C 155.7, s), C-3 (d C 136.2, s), and C-4 (d C 179.9, s), as well as those between the other hydroxy proton (d H 11.11, brs) and C-5 (d C 158.3, s), C-6 (d C 96.9, d) and C-10 (d C 109.9, s).Two doublets [7.90 (d, J 8.8, 2H) and 7.05 (d, J 8.8, 2H)] and two singlets [(d H 6.93 (s), 1H and 6.84 (s), 1H)] in the 1 H NMR spectrum also supported the substituent positions in compound 1.Thus, the structure of compound 1 was established as 3,5-dihydroxy-4'-methoxy-7,8-(2"-ethyl furan)-flavone and named as fistulaflavone A. Compound 2 was obtained as an orange gum, and showed a quasi molecular ion at m/z 391.0790 in the HRESIMS data, corresponding to the molecular formula C 20 H 16 O 7 .Comparison of 1D NMR spectra of compound 2 with those

Table 1 .
13 and13C NMR data (500 and 125 MHz, respectively) of compounds 1 and 2 in C 5 D 5 N a a Hydrogenation pattern of the carbons determined by DEPT in comparison with the HSQC spectrum.